Conventional light microscopy with its resolution of about 200 nm is inadequate to study the organization of biological molecules (e.g., proteins) whose dimensions are generally less than 10 nm. Super-resolution fluorescent microscopy methods allow one to localize the position of an isolated switchable fluorophore (fluorescent chemical compounds used to label samples of interest) by determining the center of distribution of the fluorescent photons, with an error that depends primarily on the conventional resolution divided by the square root of the number of photons collected for the fluorophore. Thus, if one collects 100 photons, one can localize an isolated fluorophore to about 20 nm. The more photons one collects, the smaller the error or the better the “resolution.”
The process of super-resolution fluorescent microscopy consists of activating (i.e., turning on the fluorescence) a limited number of photo-switchable fluorescent tags, mapping their positions and then repeating the cycle until all fluorophores have been activated and their fluorescence measured. The accuracy of localization, which depends on the number of total photons collected, depends on the probability of bleaching (i.e., destroying) the fluorophore as a function of the number of excitations. The positions of the set of sample molecules are determined from all the images and are plotted on a pseudo image thereby generating the super-resolution images.
Besides the number of photons collected, motion of the fluorophores during the repeated cycle of activation and mapping limits resolution. To achieve the highest accuracy in localization (that is, the maximum resolution), the relative motion between sample molecules must be eliminated. Although theoretical resolutions of a couple of nanometers is theoretically possible, currently only about 20 nm resolution is the norm.
Using microscopy at cryogenic temperatures, e.g., below −135° Centigrade (C) has several beneficial effects related to improving image resolution. At such temperatures, molecular motion is reduced relative to that at room temperature. Samples that are fast or high-pressure frozen preserve native structure if held below −135° C. (e.g., in amorphous ice). Furthermore, the rate of bleaching of fluorophores is greatly reduced. By decreasing temperature of observation from room temperature to −135° C., bleaching rate can be reduced by a factor of about 50, which would increase resolution by a factor of about 7.
Several different approaches have been taken for holding samples at cryo-temperatures while viewing them in a light microscope. Commercially available devices, such as those available from Instec Inc. (Boulder, Colo.) or Linkam Scientific Instruments (Guildford, Surrey, U.K.) fit on standard light microscopes, insulating the objective lens from the cold sample by a set of windows separated by air gaps. Such devices necessarily use objectives with a relatively low numerical aperture (NA) (a dimensionless number that characterizes the range of angles over which the system can accept or emit light), since the angle of transmitted light from the specimen to the objective is limited by total internal reflection at the window (i.e., coverslip) to air interface. The conventional resolution varies inversely with the NA, and the square root of the number of photons collected varies with the NA. Thus, the super-resolution varies inversely with the square of the NA. When gaps of several millimeters are required as is the case for the commercial cold stages, the highest NA lenses have a NA of about 0.4, which causes a reduction in resolution by a factor of about 9 relative to an immersion lens having a NA of about 1.2. Immersion lenses are used in standard super-resolution microscopy at room temperature and therefore set the standard. Hence the gain of a factor of about 7 achieved with a cryo-temperature is completely offset by the use of a low NA objective. If the gap is reduced to under a millimeter, higher NA lenses can be used but the loss is still a factor of about 3 to 4 relative to an immersion objective.
Other devices have used air-coupled lenses that in some cases have shortened the working distance so that lenses of higher NA (e.g., 0.75) can be used. Such systems nonetheless still result in a loss of about 60% of the photons.
Another reported attempt to provide a sample holder at cryogenic temperatures permits the sample to be held at cold temperatures while viewing it with a high NA objective by immersing the objective lens in a cryo-fluid having the approximate refractive index of water. The device has two drawbacks: it cannot be used with a conventional light microscope; and the objective must be operated at the same extremely cold temperatures. This latter condition means that the distances and dimensions designed to provide the highest performance are altered by the contraction of the parts of objective in the cold. Since the high NA objectives have cemented doublets, the objectives can fail as the glue holding the doublets together fails during repeated cycles of contraction and expansion.
Thus, there is presently no super-resolution cryogenic microscope assembly which can be used with a conventional light microscope without compromising the imaging resolution or performance of the microscope.
Any attempts to maintain a sample at cryogenic temperatures while viewing it with an objective maintained at operating temperatures that allow for standard to high performance must grapple with the larger problem of maintaining a temperature differential over a small working distance. Heat (i.e., energy transferred between a system and its surroundings other than by work or transfer of matter) flows spontaneously (e.g., via conduction, convection, and/or radiation) from a hotter system to a colder system. When two or more systems or objects, for example, a sample and a conventional light microscope, come into thermal contact, they exchange energy through the microscopic interactions of their constituent particles. When the systems are at different temperatures, the result is a spontaneous net flow of energy that continues until the systems are in thermal steady state (e.g., the temperatures are equal). The rate of heat transfer is inversely proportional to the distance between the systems (i.e., the smaller the distance, the faster the transfer). The maintenance of a steady state temperature differential between two or more systems, particularly within a relatively small working distance from each other like a microscopy sample and immersion liquid, remains a challenge.